Genes with es cell-specific expression

ABSTRACT

The invention provides an antibody that specifically binds to (a) a protein having an amino acid sequence depicted in SEQ ID NO:16 or 32 or (b) a protein which has an amino acid sequence of (a), wherein one to several amino acids are deleted, substituted, or added, and which is specifically expressed in an ES cell.

CROSS-REFERENCE TO RELATED APPLICATION

This patent application is a divisional of copending U.S. patentapplication Ser. No. 11/483,880, filed on Jul. 10, 2006, which is adivisional of U.S. patent application Ser. No. 10/479,334, filed Dec. 1,2003, issued as U.S. Pat. No. 7,250,255 on Jul. 31, 2007, which is theU.S. national phase of International Patent Application No.PCT/JP2002/05350, filed May 31, 2002, which claims the benefit ofJapanese Patent Application No. 2001-165927, filed on May 31, 2001, thecontents of which are hereby incorporated by reference.

INCORPORATION-BY-REFERENCE OF MATERIAL ELECTRONICALLY SUBMITTED

Incorporated by reference in its entirety herein is a computer-readablenucleotide/amino acid sequence listing submitted concurrently herewithand identified as follows: 158,753 bytes ASCII (Text) file named“710020SequenceListing.txt,” created Mar. 13, 2012.

TECHNICAL FIELD

The present invention relates to an ECAT gene (ES cell associatedtranscript gene) specifically expressed in ES cells (embryonic stemcells) and use thereof.

BACKGROUND ART

Embryonic stem (ES) cell is a cell isolated from early embryo of mammal,which semipermanently continues to proliferate, while maintaining anability to differentiate into any cell in the body, i.e., pluripotency.ES cell was first established in mouse in 1981, and brought anepoch-making technique of gene function analysis using knock out mice.Ever since the establishment of human ES cell was reported in 1998,application thereof to regenerative medicine has been highly expected.It is an attempt to achieve functional recovery by transplanting heartmuscle cells or nerve cells differentiated from ES cells into patientswith heart infarction and neurodegenerative diseases.

While the cell transplant therapy has been already, employed, astypically seen in marrow graft in leukemia, it is associated with twoproblems of securing sufficient supply of cells to be transplanted andsuppression of rejection reaction. Use of the ES cell that dividessemipermanently altogether solves the problem of secured supply ofsufficient amount of cell. When combined with the somatic cell clonetechnology, moreover, the rejection reaction can be also overcome. Whenan ES cell is established from a clone embryo prepared from the somaticcell of a patient and used for transplantation, rejection cannot occursince it has the same gene as does the patient. Therefore, ES cell hasthe potential to simultaneously solve the two problems in the celltransplant therapy.

While ES cell has the high potential as described above, human ES cellis difficult to establish and maintain as compared to mouse ES cell.Therefore, the development of a reliable establishment technique and aculture technique is necessary. For a human ES cell to be established,moreover, an embryo needs to be sacrificed. When it is combined with thesomatic cell clone technology, it easily leads to human cloning. Tosolve such ethical issues, therefore, the development of a technique todirectly produce an ES-like cell having pluripotency from a somatic cellis desired, which does not go through an embryo.

What plays a key role in the development of these techniques is a gene(ES cell associated transcript gene, hereinafter ECAT gene), which isspecifically expressed in pluripotent cells such as ES cell and thelike. The ECAT gene becomes a marker to determine if the cell is an EScell. In addition, ES cell can be efficiently selected from a mixedculture of various kinds of cells by combining a control region of ECATgene that induces ES cell specific expression and a drug resistance gene(JP-T-9-500004; corresponding U.S. Pat. No. 6,146,888). Furthermore, itmay be possible to promote conversion of somatic cell to ES-like cell byinducing expression of SCAT gene.

The only one gene reported heretofore as an ECAT gene is transcriptionfactor Oct3 (also called Oct4, POU5f1, hereinafter to be referred to asOct-3/4) gene. While a similar gene has been reported with regard tohuman (hereinafter to be referred to as hOct-3/4 gene: Takeda et al.,Nucleic Acids Res. 20: 4613-4620, 1992, SEQ ID NO: 39), no report hasbeen so far found on verified ES cell specific expression of hOct-3/4gene. Oct-3/4 is a transcription factor that is specifically expressedin an ES cell and EG cell (embryonic germ cells), whose expressiondisappears along with the cell differentiation. Therefore, it is used asa marker of ES cell, and efficient establishment of ES cell has beenattempted by knocking-in a neomycin resistance gene into its gene locus(JP-T-9-500004; corresponding to U.S. Pat. No. 6,146,888). However, areport has also documented that Oct-3/4 is expressed in trophectodermcell as well, besides pluripotent cells (Biol Reprod 63: 1698-1705,2000). Thus, use of Oct-3/4 gene alone as an index results in theselection of cells other than ES cells. To avoid this risk, it isdesirable to identify plural ECAT genes and use them in combination.Even if expression of Oct-3/4 alone in somatic cell is induced,conversion to ES-like cell is not observed. Even if Oct-3/4 isconstantly expressed, differentiation of ES cell (differentiation intoprimitive-endoderm, primitive ectoderm) associated withdrawal of LIF(leukemia inhibitor factor) cannot be suppressed. To the contrary, aninteresting report has been made that, by increasing the expressionamount of Oct-3/4 by only about 1.5 times the general level,differentiation similar to that associated with the withdrawal of LIF isinduced (Experimental Medicine, 19, 330-338, 2001). As described above,the action of Oct-3/4 is not simple and induction thereof into ES cellby the expression of Oct-3/4 alone in somatic cell is difficult. Fromthis aspect, too, it is considered necessary to combine plural SCATgenes and analyze ES cell.

Nevertheless, SCAT gene other than Oct-3/4 gene has not been found andthere is a strong demand for the provision of a new SCAT gene, from theaspects of regenerative medicine and application of ES cells to celltransplantation.

DISCLOSURE OF THE INVENTION

The present invention aims at provision of a novel SCAT gene. Moreparticularly, the present invention aims at provision of a screeningmethod of ES cell using the new ECAT gene and a gene product peptideencoded thereby, as well as a probe for selecting an ES cell.

To identify SCAT candidate genes, the present inventors used the EST(Expressed Sequence Tag) data base (detail to be described later) forcomputer analysis and identified candidate genes to reach 10 genes. Ofthe 10 genes, 8 genes were subjected to Northern blotting, wherebyexpression in ES cell and 12 kinds of organs (mouse) was analyzed. As aresult, the expression of all the 8 genes was found to be specific to EScells. It was also found that the expression of these genes quicklydisappeared after stimulation of ES cell with retinoic acid, namely, byinduction of differentiation. From the above results, the presentinventors have found that these 8 genes are SCAT genes, which resultedin the completion of the present invention. Of the remaining two genes,one gene was analyzed by Northern blotting and the like to find the geneto be an ECAT gene.

Further, they have identified a human gene homologous to the ECAT gene(hereinafter hECAT) and analyzed expression in the ES cell and 13 kindsof organs (human).

Accordingly, the present invention provides the following.

(1) A probe for selecting ES cells, comprising a DNA which has a basesequence depicted in any one of SEQ ID NOs: 1, 3, 4, 5, 6, 7 and 8.(2) A probe for selecting ES cells, comprising a DNA which hybridizes toa DNA having a base sequence depicted in any one of SEQ ID NOs: 1, 3, 4,5, 6, 7 and 8 under stringent conditions, and which encodes a proteinspecifically expressed in an ES cell.(3) A probe for selecting ES cells, comprising a DNA which has a basesequence depicted in SEQ ID NO: 1, 3, 4, 5, 6, 7 or 8, wherein one toseveral bases are deleted, substituted or added, and which is capable ofhybridizing, under stringent conditions, to a DNA encoding a proteinspecifically expressed in an ES cell.(4) A probe for selecting ES cells, comprising a DNA which has a basesequence depicted in any one of SEQ ID NOs: 9, 13, 15, 17, 19, 21, 23and 41.(5) The probe of the above-mentioned (4), comprising a DNA which has abase sequence depicted in any one of SEQ ID NOs: 9, 13, 15, 17, 19, 21and 23.(6) A probe for selecting ES cells, comprising a DNA which hybridizes toa DNA having a base sequence depicted in any one of SEQ ID NOs: 9, 13,15, 17, 19, 21, 23 and 41 under stringent conditions, and which encodesa protein specifically expressed in an ES cell.(7) The probe of the above-mentioned (6), comprising a DNA whichhybridizes to a DNA which has a base sequence depicted in any one of SEQID NOs: 9, 13, 15, 17, 19, 21 and 23 under stringent conditions, andwhich encodes a protein specifically expressed in an ES cell.(8) A probe for selecting ES cells, comprising a DNA which has a basesequence depicted in SEQ ID NO: 9, 13, 15, 17, 19, 21, 23% or 41,wherein one to several bases are deleted, substituted or added, andwhich is capable of hybridizing, under stringent conditions, to a DNAencoding a protein specifically expressed in an ES cell.(9) The probe of the above-mentioned (8), comprising a DNA which has abase sequence depicted in SEQ ID NO: 9, 13, 15, 17, 19, 21 or 23,wherein one to several bases are deleted, substituted or added, andwhich is capable of hybridizing, under stringent conditions, to a DNAencoding a protein specifically expressed in an ES cell.(10) The probe of any of the above-mentioned (1) to (9), which is usedfor selecting a mouse ES cell.(11) A probe for selecting ES cells, comprising a DNA which has a basesequence depicted in any one of SEQ ID NOs: 27, 29, 31, 33, 35, 37 and43.(12) The probe of the above-mentioned (11), which comprises a DNA whichhas a base sequence depicted in any one of SEQ ID NOs: 27, 29, 31, 33,35 and 37.(13) A probe for selecting ES cells, comprising a DNA which hybridizesto a DNA which has a base sequence depicted in any one of SEQ ID NOs:27, 29, 31, 33, 35, 37 and 43 under stringent conditions, and whichencodes a protein specifically expressed in an ES cell.(14) The probe of the above-mentioned (13), which comprises a DNA whichhybridizes to a DNA which has a base sequence depicted in any one of SEQID NOs: 27, 29, 31, 33, 35 and 37 under stringent conditions, and whichencodes a protein specifically expressed in an ES cell.(15) A probe for selecting ES cells, comprising a DNA which has a basesequence depicted in SEQ ID NO: 27, 29, 31, 33, 35, 37 or 43, whereinone to several bases are deleted, substituted or added, and which iscapable of hybridizing, under stringent conditions, to a DNA encoding aprotein specifically expressed in an ES cell.(16) The probe of the above-mentioned (15), comprising a DNA which has abase sequence depicted in SEQ ID NO: 27, 29, 31, 33, or 37, wherein oneto several bases are deleted, substituted or added, and which is capableof hybridizing, under stringent conditions, to a DNA encoding a proteinspecifically expressed in an ES cell.(17) The probe of any of the above-mentioned (11) to (16), which is usedfor selecting a human ES cell.(18) A gene comprising a DNA of any of the following (a)-(c):(a) a DNA comprising a base sequence depicted in SEQ ID NO: 17(b) a DNA which hybridizes to a DNA having a base sequence of (a) understringent conditions, which encodes a protein specifically expressed inan ES cell(c) a DNA which has a base sequence of (a), wherein one to several basesare deleted, substituted or added, and which is capable of hybridizing,under stringent conditions, to a DNA encoding a protein specificallyexpressed in an ES cell.(19) A protein of the following (a) or (b):(a) a protein having an amino acid sequence depicted in SEQ ID NO: 18(b) a protein which has an amino acid sequence of (a), wherein one toseveral bases are deleted, substituted or added, and which isspecifically expressed in an ES cell.(20) A gene comprising a DNA of any of the following (a)-(c):(a) a DNA comprising a base sequence depicted in SEQ ID NO: 29(b) a DNA which hybridizes to a DNA having a base sequence of (a) understringent conditions, and which encodes a protein specifically expressedin an ES cell(c) a DNA which has a base sequence of (a), wherein one to several basesare deleted, substituted or added, and which is capable of hybridizing,under stringent conditions, to a DNA encoding a protein specificallyexpressed in an ES cell.(21) A protein of the following (a) or (b):(a) a protein having an amino acid sequence depicted in SEQ ID NO: 30(b) a protein which has an amino acid sequence of (a), wherein one toseveral bases are deleted, substituted or added, and which isspecifically expressed in an ES cell.(22) A gene comprising a DNA of any of the following (a)-(c):(a) a DNA comprising a base sequence depicted in SEQ ID NO: 33(b) a DNA which hybridizes to a DNA having a base sequence of (a) understringent conditions, and which encodes a protein specifically expressedin an ES cell(c) a DNA which has a base sequence of (a), wherein one to several basesare deleted, substituted or added, and which is capable of hybridizing,under stringent conditions, to a DNA encoding a protein specificallyexpressed in an ES cell.(23) A protein of the following (a) or (b):(a) a protein having an amino acid sequence depicted in SEQ ID NO: 34(b) a protein which has an amino acid sequence of (a), wherein one toseveral bases are deleted, substituted or added, and which isspecifically expressed in an ES cell.(24) A gene comprising a DNA of any of the following (a)-(c):(a) a DNA comprising a base sequence depicted in SEQ ID NO: 37(b) a DNA which hybridizes to a DNA having a base sequence of (a) understringent conditions, and which encodes a protein specifically expressedin an ES cell(c) a DNA which has a base sequence of (a), wherein one to several basesare deleted, substituted or added, and which is capable of hybridizing,under stringent conditions, to a DNA encoding a protein specificallyexpressed in an ES cell.(25) A protein of the following (a) or (b):(a) a protein having an amino acid sequence depicted in SEQ ID NO: 38(b) a protein which has an amino acid sequence of (a), wherein one toseveral bases are deleted, substituted or added, and which isspecifically expressed in an ES cell.(26) A method of screening an ES cell, which comprises analyzing anintracellular expression state of a DNA having a base sequence depictedin SEQ ID NO: 9, 13, 15, 17, 19, 21, 23 or 41, or a protein having anamino acid sequence depicted in SEQ ID NO: 10, 14, 16, 18, 20, 22, 24 or42.(27) The method of the above-mentioned (26), wherein the intracellularexpression state of a DNA having a base sequence depicted in SEQ ID NO:9, 13, 15, 17, 19, 21 or 23, or a protein having an amino acid sequencedepicted in SEQ ID NO: 10, 14, 16, 18, 20, 22 or 24 is analyzed.(28) The method of the above-mentioned (26) or (27), which furthercomprises analyzing an intracellular expression state of a DNA having abase sequence depicted in SEQ ID NO: 11, or a protein having an aminoacid sequence depicted in SEQ ID NO: 12.(29) The method of any of the above-mentioned (26) to (28), whichfurther comprises analyzing an intracellular expression state of a DNAhaving a base sequence depicted in SEQ ID NO: 25, or a protein having anamino acid sequence depicted in SEQ ID NO: 26.(30) A method of screening an ES cell, which comprises analyzing anintracellular expression state of a DNA having a base sequence depictedin SEQ ID NO: 27, 29, 31, 33, 35, 37 or 43, or a protein having an aminoacid sequence depicted in SEQ ID NO: 28, 30, 32, 34, 36, 38 or 44.(31) The method of the above-mentioned (30), wherein the intracellularexpression state of a DNA having a base sequence depicted in SEQ ID NO:27, 29, 31, 33, 35 or 37, or a protein having an amino acid sequencedepicted in SEQ ID NO: 28, 30, 32, 34, 36 or 38 is analyzed.(32) The method of the above-mentioned (30) or (31), which furthercomprises analyzing an intracellular expression state of a DNA having abase sequence depicted in SEQ ID NO: 39, or a protein having an aminoacid sequence depicted in SEQ ID NO: 40.(33) A probe for selecting ES cells, comprising a DNA which has anon-repetitive sequence comprising not less than 20 continuous basesfrom a base sequence depicted in SEQ ID NO: 9, 11, 13, 15, 17, 19, 21,23 or 41, or SEQ ID NO: 27, 29, 31, 33, 35, 37 or 43, and which has asequence specific to a gene specifically expressed in an ES cell.(34) A method of screening an ES cell, which comprises analyzing anexpression state of a gene specifically expressed in an ES cell, using aprobe of any of the above-mentioned (1)-(17) and (33).(35) The method of the above-mentioned (34), which further comprisesusing a probe for selecting ES cells comprising a DNA having a basesequence depicted in SEQ ID NO: 2 or 11.(36) The method of the above-mentioned (34) or (35), which furthercomprises using an ES cell selection probe comprising a DNA having abase sequence depicted in SEQ ID NO: 25.(37) The method of the above-mentioned (34), which further comprisesusing an ES cell selection probe comprising a DNA having a base sequencedepicted in SEQ ID NO: 27.(38) The method of the above-mentioned (34) or (35), which furthercomprises using an ES cell selection probe comprising a DNA having abase sequence depicted in SEQ ID NO: 39.

The present invention further relates to a recombinant vector having aDNA encoding a gene specifically expressed in an ES cell or a proteinspecifically expressed in an ES cell, particularly, a vector for forcedexpression of a differentiation inhibiting gene (pluripotency sustaininggene), and a transformant cell transformed with said vector.

The present invention moreover relates to a recombinant vectorcomprising a selection gene such as a drug resistance gene or the like,which is incorporated into a genomic DNA fragment containing a DNAencoding a gene specifically expressed in an ES cell or a proteinspecifically expressed in an ES cell, particularly a vector forselecting ES cell, and a transformant cell transformed with said vector.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows an analysis of the expression of each ECAT gene in an EScell and 12 kinds of organs in adult mouse by Northern blotting.

FIG. 2 shows an analysis of the expression of each ECAT gene in an EScell, a mesenchymal stem cell and 13 kinds of organs in adult human byNorthern blotting.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a gene specifically expressed in an EScell (hereinafter to be also referred to as gene with ES cell-specificexpression), namely, ECAT gene. It is possible to determine if a cell isan ES cell with the presence of expression of ECAT gene as an index. Thepresent invention provides an ES cell selection probe preferable fordeciding on the ES cell as described. As this probe, a polynucleotidecontaining a DNA comprising a base sequence depicted in any one of SEQID NOs: 1-8, a DNA comprising a base sequence depicted in SEQ ID NO: 9(hereinafter ECAT1 gene), a DNA comprising a base sequence depicted inSEQ ID NO: 11 (hereinafter ECAT2 gene), a DNA comprising a base sequencedepicted in SEQ ID NO: 13 (hereinafter ECAT3 gene), a DNA comprising abase sequence depicted in SEQ ID NO: 15 (hereinafter ECAT4 gene), a DNAcomprising a base sequence depicted in SEQ ID NO: 17 (hereinafter ECAT5gene), a DNA comprising a base sequence depicted in SEQ ID NO: 19(hereinafter ECAT6 gene), a DNA comprising a base sequence depicted inSEQ ID NO: 21 (hereinafter ECAT7 gene), a DNA comprising a base sequencedepicted in SEQ ID NO: 23 (hereinafter ECAT8 gene) or a DNA comprising abase sequence depicted in SEQ ID NO: 41 (hereinafter ECAT9 gene) can bespecifically mentioned. In the present invention, moreover, the ES cellselection probe may be any as long as it can achieve the object ofconfirmation of the presence or otherwise of the expression of ECATgene, and may be the above-mentioned base sequence which underwentmodification by substitution, deletion, addition and the like.Specifically, a polynucleotide comprising a DNA that hybridizes to anECAT gene under stringent conditions and encodes a protein specificallyexpressed in an ES cell, and a polynucleotide comprising a DNA which hasa base sequence of an ECAT gene, wherein one to several bases aredeleted, substituted or added, and which is capable of hybridizing,under stringent conditions, to a DNA encoding a protein specificallyexpressed in an ES cell, can be preferably used as a probe for selectingES cells in the present invention. Specific examples include apolynucleotide containing a DNA comprising a base sequence depicted inSEQ ID NO: 27 (hereinafter hECAT2 gene), a DNA comprising a basesequence depicted in SEQ ID NO: 29 (hereinafter hECAT3 gene), a DNAcomprising a base sequence depicted in SEQ ID NO: 31 (hereinafter hECAT4gene), a DNA comprising a base sequence depicted in SEQ ID NO: 33(hereinafter hECAT5 gene), a DNA comprising a base sequence depicted inSEQ ID NO: 35 (hereinafter hECAT7 gene), a DNA comprising a basesequence depicted in SEQ ID NO: 37 (hereinafter hECAT8 gene) or a DNAcomprising a base sequence depicted in SEQ ID NO: 43 (hereinafter hECAT9gene).

In addition, a polynucleotide containing a DNA comprising a basesequence depicted in SEQ ID NO: 25, i.e., a DNA encoding Oct-3/4, or aDNA comprising a base sequence depicted in SEQ ID NO: 39, i.e., a DNAencoding hOct-3/4 can be also used as an ES cell selection probe.Because a report has documented that the Oct-3/4 gene is expressed evenin trophectoderm cells as mentioned above, concurrent use of apolynucleotide preferably containing an ECAT gene other than Oct-3/4gene or hOct-3/4 gene and the like, such as the novel ES cell selectionprobe of the present invention is preferable. Even in the case of anovel ES cell selection probe containing the above-mentioned ECAT gene,concurrent use of several kinds of probes is preferable to moreaccurately determine if it is an ES cell.

In the present specification, the term “stringent conditions” means theconditions under which a DNA having about 70% or more, preferably about80% or more, particularly preferably about 90% or more, homology in abase sequence can hybridize, wherein stringency can be controlled byappropriately changing the temperature, salt concentration and the likeduring hybridizing reaction and washing. More preferable conditions arethose under which a DNA having about not less than 95% homology canhybridize.

ECAT2 gene is reported as a gene pH34 that shows a decreased expressionwhen EC cell is stimulated with retinoic acid (Differentiation 46:61-67, 1991), and according to the database of RIKEN, it is described asESG (ES cell specific gene) 1. Furthermore, ECAT3 gene is a geneencoding a mouse protein having an F-box, whose expression in orchis andovary is reported (Current Biology 9: 1180-1182, 1999). ECAT7 gene isreported as protein DNMT3L that is similar to DNMT3 that causes DNAmethylation (Genomics 65: 293-298, 2000). ECAT9 gene is reported as agrowth factor called GDF3, in Jones CM et al., Mol Endocrinol. 6:1961-1968, 1992 for mouse and in Caricasole et al., Oncogene 16: 95-103,1998 for human. There is no report on an ES cell specific expression.With regard to ECAT4 gene, ECAT5 gene and ECAT6 gene, no report is foundin published literatures, but by a protein database search has revealedthat ECAT4 gene has a homeo box, ECAT5 gene has homology with oncogeneH-Ras, and that ECAT6 gene is similar to keratin. For ECAT5 gene, eventhough its partial sequence is known, a cDNA sequence per se and theamino acid sequence of a protein that the DNA sequence codes for havenot been determined. Accordingly, the present invention provides anECAT5 gene, an ECAT5 protein, a gene having extremely high homology withthese and a protein showing similar behavior with these.

As used herein, the “gene having extremely high homology” specificallymeans a gene that hybridizes to ECAT5 gene under stringent conditions,and as long as this requirement is satisfied, one to several bases maybe deleted, substituted or added in the base sequence (SEQ ID NO: 17) ofECAT5 gene. Specifically, it is a gene having about 70% or more,preferably about 80% or more, more preferably about 90% or more,particularly preferably 95% or more, homology with the ECAT5 gene. The“protein showing similar behavior” means a protein having thecharacteristics that the ECAT5 protein shows, namely, being specificallyexpressed in an ES cell. As long as this requirement is satisfied, oneto several amino acids may be deleted, substituted or added in the aminoacid sequence (SEQ ID NO: 18) of ECAT5 protein.

Moreover, the ES cell selection probe of the present inventionencompasses a DNA fragment consisting of a partial is sequencecomprising 20 or more continuous bases without a repeated sequence, fromthe base sequence described in SEQ ID NO: 9, 11, 13, 15, 17, 19, 21, 23or 41, or SEQ ID NO: 27, 29, 31, 33, 35, 37 or 43, which are constructedbased on the sequences of various ECAT genes and hECAT genes. The DNAfragment is not particularly limited as long as it can hybridize to SCATgene or hECAT gene. Specifically, it is a DNA containing a continuouspartial sequence generally comprising 20 bases or more, preferably about100 bases or more, and more preferably about 200 bases or more, of thebase sequence of each SEQ ID No, which contains at least a sequencespecific to various ECAT genes or hECAT genes intended for detection,and which does not consist of a repeated sequence alone. Preferableexamples thereof include a DNA fragment depicted in SEQ ID NOs: 1-8.

Of the aforementioned 9 kinds of mouse ECAT genes, 7 kinds of ECAT2gene, ECAT3 gene, ECAT4 gene, ECAT5 gene, ECAT7 gene, ECAT8 gene andECAT9 gene have been found to have the corresponding human ECAT genes(mentioned below: hECAT2 gene, hECAT3 gene, hECAT4 gene, hECAT5 gene,hECAT7 gene, hECAT8 gene and hECAT9 gene, respectively). Of these, thebase sequences of hECAT3, hECAT5 and hECAT8 genes and the amino acidsequences of the proteins encoded by the base sequences have not beendetermined. Accordingly, the present invention provides the genes andproteins of hECAT3, hECAT5 and hECAT8, as well as genes having extremelyhigh homology therewith and proteins showing similar behaviors.

Here, the “genes having extremely high homology” and the “proteinsshowing similar behaviors” specifically mean genes that hybridize to thegenes of hECAT3, hECAT5 or hECAT8 under stringent conditions. As long asthis requirement is satisfied, one to several bases may be deleted,substituted or added in the base sequences of hECAT3, hECAT5 and hECAT8genes (SEQ ID NO: 29, SEQ ID NO: 33 and SEQ ID NO: 37, respectively). Tobe precise, it is a gene having about 70% or more, preferably about 80%or more, more preferably about 90% or more, particularly preferably 95%or more, homology with those genes. The “proteins showing similarbehaviors” mean proteins having the characteristics of the hECAT3protein, hECAT5 protein or hECAT8 protein. As long as this requirementis satisfied, one to several amino acids may be deleted, substituted oradded in the amino acid sequences of hECAT3 protein, hECAT5 protein andhECAT8 protein (SEQ ID NO: 30, SEQ ID NO: 34 and SEQ ID NO: 38,respectively).

The probe of the present invention can be prepared according to themethods known in this field. For example, this probe can be prepared asa DNA isolated by cleaving EST of the corresponding ECAT gene with arestriction enzyme, a DNA obtained by amplification of PCR using, as atemplate, genomic DNA, complementary DNA (cDNA) prepared from EScell-derived mRNA, chemically synthesized DNA, and a DNA constructed bya suitable combination of these methods.

The present invention provides a screening method of ES cell, which ischaracterized by analyzing the expression state of a gene specificallyexpressed in an ES cell. As used herein, the “gene specificallyexpressed in an ES cell” is the same as the aforementioned ECAT gene orhECAT gene, and is specifically exemplified by ECAT1 gene, ECAT2 gene,ECAT3 gene, ECAT4 gene, ECAT5 gene, ECAT6 gene, ECAT7 gene, ECAT8 gene,ECAT9 gene, hECAT2 gene, hECAT3 gene, hECAT4 gene, hECAT5 gene, hECAT7gene, hECAT8 gene and hECAT9 gene, as well as Oct-3/4gene, hOct-3/4 geneand the like.

In the present invention, ES cell is screened by analyzing theexpression state of a gene specifically expressed in ES cells or aprotein specifically expressed in ES cells and encoded by said gene. Forthe analysis of expression state at the gene level, the aforementionedprobe for selecting ES cells can be used. It is also preferable toconcurrently use a probe comprising a polynucleotide having a DNAencoding Oct-3/4, as mentioned above. Such probe may be labeled with afluorescent substance, an enzyme, a radioisotope or the like. For theanalysis of the expression state at the protein level, a substancehaving specific affinity for the above-mentioned protein specificallyexpressed in ES cells, such as an antibody, is used to examineintracellular expression of the protein. More specifically, methodsutilizing an antigen-antibody reaction generally practiced in thepertinent field, such as immunoblot, immunoprecipitation and the like,are used. The antibody here is not particularly limited as long as itcan specifically bind to the protein, and may be any of a polyclonalantibody, a monoclonal antibody and a functional fragment thereof. Theseantibodies and fragments thereof may be labeled with a fluorescentsubstance, an enzyme, a radioisotope or the like.

Moreover, they may be commercially available ones or may be preparedappropriately according to a conventional method.

The present invention relates to an expression vector comprising any ofthe above-mentioned gene specifically expressed in ES cells and a geneencoding a protein specifically expressed in ES cells. As used herein,the gene specifically expressed in ES cells is as defined above, and thegene encoding a protein specifically expressed in ES cells isspecifically a gene encoding ECAT1 (SEQ ID NO: 10), a gene encodingECAT2 (SEQ ID NO: 12) or hECAT2 (SEQ ID NO: 28), a gene encoding ECAT3(SEQ ID NO: 14) or hECAT3 (SEQ ID NO: 30), a gene encoding ECAT4 (SEQ IDNO: 16) or hECAT4 (SEQ ID NO: 32), a gene encoding ECAT5 (SEQ ID NO: 18)or hECAT5 (SEQ ID NO: 34), a gene encoding ECAT6 (SEQ ID NO: 20), a geneencoding ECAT7 (SEQ ID NO: 22) or hECAT7 (SEQ ID NO: 36), a geneencoding ECAT8 (SEQ ID NO: 24) or hECAT8 (SEQ ID NO: 38) and a geneencoding ECAT9 (SEQ ID NO: 42) or hECAT9 (SEQ ID NO: 44) can bementioned. The expression vector preferably has a function ofsuppressing the differentiation by expression of the vector in the cell,particularly ES cells, in light of the nature of the gene contained inthe vector. In other words, it is a vector that forcibly expresses adifferentiation inhibiting gene (pluripotency sustaining gene)(hereinafter to be also referred to as vector for forced expression ofthe differentiation inhibiting gene (pluripotency sustaining gene)). Theexpression vector of the present invention is not particularly limitedas long as it is capable of maintaining replicability or autonomousgrowth in various animal cells and expressing the gene specificallyexpressed in ES cells, and encompasses virus vector, plasmid vector andthe like. This expression vector can be prepared based on conventionalgenetic engineering, for example, according to basic textbooks such asMolecular cloning 2nd Ed., Cold Spring Harbor Laboratory Press (1989)and the like. The vector is preferably a virus vector, which is preparedby incorporating gene specifically expressed in ES cells or the likeinto DNA virus or RNA virus such as retrovirus, adenovirus,adeno-associated virus, herpesvirus, vaccinia virus, poxvirus,poliovirus, sindbis virus or the like. Where necessary, a desiredpromoter region, a drug resistance gene region or an expressionregulatory region can be also introduced.

The expression vector of the present invention is introduced into a cellaccording to conventionally known methods such as transfection,lipofection, microinjection, gene gun, electroporation or the like.

Whether or not the expression vector of the present invention thusprepared is incorporated into a host cell and expressed can be confirmedby, for example, determining the amount of protein (polypeptide) thatthe introduced ECAT gene expressed and produced by, for example, ELISAand the like.

In addition to the use of the ECAT gene as a probe to determine if acell is an ES cell, the ECAT gene can be also used for selectiveseparation of ES cells from a mixture of ES cells and other kinds ofcells. The present inventors have prepared a targeting vector toknock-in a drug selection gene into a protein translation region of eachECAT gene and, using this vector, established ES cells that causedhomologous recombination. Specifically, the technique described inJP-T-9-500004 (corresponding to U.S. Pat. No. 6,146,888) was applied.For example, a cell wherein a neomycin resistance gene had beenknocked-in into an ECAT3 gene, ECAT4 gene or ECAT5 gene was cultured inthe presence of G418, but cell differentiation was not observed in aselected cell. Such results suggest a possible use of the SCAT gene forthe selective separation of ES cells. For an ensured selection of EScells alone, it is preferable to perform homologous recombination usingplural kinds of vectors incorporating different ECAT genes.

EXAMPLES

The present invention is explained in detail by referring to Examples,which are not to be construed as limitative.

Example 1 Identification of Mouse SCAT Gene (1) Identification ofCandidate Gene by Computer Analysis (Procedure)

The EST database was used to identify the candidate genes of SCAT. ESTis obtained by randomly extracting a number of cDNA clones from cDNAlibraries derived from various cells and organs, analyzing only onereaction of the 5′ or 3′ end sequence thereof and registering same in apublic database. ESTs can be said to be a catalog of genes expressed ineach cell and each organ. More than one million clones derived frommouse and more than 30000 clones derived from mouse ES cell have beenregistered.

As the EST database, Unigene was used. Unigene is prepared by clusteringclones of EST, which are considered to be derived from the same gene,and as of Mar. 5, 2001, 79917 sets have been reported for the mouse ESTdatabase, where each set consists of at least one EST or known gene.

As a method of analysis, Digital differential display method was used.This method is used to examine the frequency of presence of each set inthe libraries of designated cells and organs, namely, the number of ESTclones contained in each set is divided by the number of entire ESTregistrations derived from the library thereof, thereby to examine thefrequency of expression between different cells and between differentorgans.

The frequency of gene expression in the libraries derived from thefollowing 5 kinds of cells and an organ was analyzed by the Digitaldifferential display method. The number in the parenthesis for eachgroup is the number of analyzed clones. For Group 1 to Group 5, all thecorresponding libraries were analyzed. Since the data of Group 6contained enormous quantity, 23 libraries extracted while includingorgans and cells of the entire body as many kinds as possible wereanalyzed.

Group 1 fertilized eggs from 1-cell stage to blastocyst (49050 clones)Group 2 ES cell or Embryonic carcinoma cells (32277 clones)Group 3 fetus up to 8.5 days after fertilization (46728 clones)Group 4 fetus after 9 days from fertilization (128882 clones)Group 5 orchis (65685 clones)Group 6 other cells, tissues (272460 clones)

As regards the set expected to specifically express in fertilized eggsand pluripotent cells, such as ES cell and the like, by the Digitaldifferential display method, the mouse-derived EST database (UnigeneMouse Sequence Collection) was searched using BlastN to examine if ESTwas present only in the pluripotent cell-derived libraries.

As a result of the analysis by the Digital differential display methodand EST database search using BlastN, 10 genes were identified. ESTs ofthese genes were highly frequently present in fertilized eggs and EScells but were not found in other cells and tissues of Group 6. WhileEST was included in the fetus- and orchis-derived libraries for somegenes, since this was highly likely derived from primordial germ cell orsperm mother cell, which is a pluripotent cell, they were included inthe candidates for ECAT gene. While Oct-3/4 gene was present at highfrequency in the fertilized eggs and ES cell, it was also contained inother cells and organs, though small in number. Of the candidates,mouse-derived EST database was searched for 8 genes using BlastN, theresults of which are shown in Table 1 (ECAT1-8).

Of the remaining two genes, one gene (ECAT9) was analyzed in the samemanner. The results are shown in Table 1.

TABLE 1 EST eggs ES (EC) -E8.5 E9- testis others ECATs Group 1 Group 2Group 3 Group 4 Group 5 Group 6 Oct3/4 10 13 4 1 0 2 1 7 24 0 0 0 0 2 3218 0 0 0 0 3 37 13 0 0 0 0 4 2 14 1 1 3 0 5 0 11 0 0 0 0 6 0 7 0 0 0 0 74 9 0 0 1 2 8 0 7 0 0 2 0 9 4 11 0 0 0 2

(2) Northern Blotting Analysis (Procedure)

The candidate genes identified by computer analysis were analyzed foractual ES cell specific expression by Northern blotting. Using Trizol(manufactured by Lifetech Oriental Co. Ltd.), total RNA was extractedfrom undifferentiated ES cells and ES cells differentiation-induced withretinoic acids for 5 days. RNAs derived from various organs of adultmice were purchased from Sawady Technology Co., Ltd. The total RNA (5μg) was separated by formalin agarose gel, transferred to a nylonmembrane and fixed with UV closslink. When EST of an object gene wasavailable for purchase, this DNA was used as a probe. When EST was notavailable, a DNA fragment specific for each ECAT was amplified by PCRand used as a probe. To be specific, using the following probes, theexpression of Oct-3/4, ECAT1, ECAT2, ECAT3, ECAT4, ECAT5, ECAT6, ECAT7and ECAT8 was examined. In addition, the expression of ECAT9 was alsoexamined.

Oct-3/4: a DNA fragment containing a sequence depicted in SEQ ID NO: 25,which was prepared by cleaving plasmid Cl in BS KS (Cell 60: 461-472,1990) with EcoRI.ECAT1: a DNA fragment containing a sequence depicted in SEQ ID NO: 1,which was prepared by cleaving Mm.31054EST (#AI467128) with SalI/NotI.ECAT2: a DNA fragment containing a sequence depicted in SEQ ID NO: 2,which was prepared by cleaving pH34EST (#AA473366) with SalI/NotI.ECAT3: a DNA fragment containing a sequence depicted in SEQ ID NO: 3,which was prepared by cleaving FBX15EST(#AA571680) with SalI/NotI.ECAT4: a DNA fragment containing a sequence depicted in SEQ ID NO: 4,which was prepared by cleaving the fragment with EcoRI from a plasmidobtained by amplifying a homeobox coding region for gateway by PCR andTA cloning the same.ECAT5: a DNA fragment containing a sequence depicted in SEQ ID NO: 5,which was prepared by cleaving the fragment with EcoRI from a plasmidobtained by RT-PCR of E-RasS118/RACE11 and TA cloning.ECAT6: a DNA fragment containing a sequence depicted in SEQ ID NO: 6,which is a keratin-E PCR product (48927S/48927AS).ECAT7: a DNA fragment containing a sequence depicted in SEQ ID NO: 7,which was prepared by cleaving out from DNMT3LEST clone (AA895770,pBSSK-dnmt31) with EcoRI/XhoI.ECAT8: a DNA fragment containing a sequence depicted in SEQ ID NO: 8,which was prepared by cleaving Mm.77010RACE product from TA clonedplasmid with EcoRI.ECAT9: a DNA fragment containing a sequence depicted in SEQ ID NO: 41,which was prepared by reference to GDF3 (Jones CM. et al., mentionedabove).

Probes were labeled with ³²P-dCTP using a Mega prime DNA labeling systemmanufactured by Amersham Pharmacia.

Hybridization was performed using Quickhyb of Funakoshi Co., Ltd.Signals after washing were analyzed using BAS5000 of Fuji Photo FilmCo., Ltd.

(Results)

Of the 10 genes identified by the computer search, 9 genes were so farsubjected to Northern blotting, and the expression in ES cell and 12kinds of organs was analyzed. To be precise, the expression of each ECATgene in ES cell and 12 kinds of organs of adult mouse was each analyzedby Northern blotting, the results of which are shown in FIG. 1.

It was found that every expression relating to 9 genes was specific toES cell. While expression was somewhat observed in orchis, it wasconsidered to have been derived from sperm mother cell. It was alsofound that the expression of these genes disappear quickly when ES cellwas induced with retinoic acid stimulation. From these results, the 9genes were considered to be SCAT genes.

(3) Analysis of SCAT Gene

when ECAT gene is an unknown gene, the full length cDNA was identifiedaccording to RACE (Rapid Amplification of cDNA Ends) method using 5′RACEsystem, version 2 of Lifetech Oriental Co. Ltd. The RIKEN database ofmouse full length cDNA was searched at URL(http://genome.gsc.riken.go.jp/).

Example 2 Analysis of Known Information of the Obtained SCAT Gene (1)Blast Search

EST sequence of 8 genes confirmed to be ECAT genes as a result ofNorthern blotting was searched using Blast. As a result, the sequencesof 3 genes were already reported in papers. ECAT2 gene was reported asgene pH34 that shows a decrease in expression when EC cell is stimulatedwith retinoic acid. ECAT3 gene was reported as a mouse protein having Fbox, whose expression is observed only in orchis and ovary. ECAT7 genewas reported as protein DNMT3L similar to DNMT3 that performs DNAmethylation. Identification of full length cDNA was tried by the RACEmethod and translation region was identified for ECAT4 gene, ECAT5 geneand ECAT6 gene. Deducible amino acid sequence was searched using BlastPand it was found that ECAT4 gene has homeobox, ECAT5 gene has homologywith cancer gene H-Ras, and ECAT6 gene is similar to keratin. Inaddition, ECAT9 gene, which was newly confirmed to be SCAT gene, wasfound to be a growth factor called GDF3.

(2) Search through RIKEN Mouse cDNA Database

The mouse full length cDNA database published from in February 2001 byRIKEN was searched. As a result, full length cDNAs of 8 genes exceptECAT5 gene were found to have been published. ECAT5 gene was notincluded in the database. In addition, ECAT2 gene is described as an EScell specific gene (ESG) 1 in the RIKEN database, but no information wasavailable as regards the expression of other 8 genes in ES cell.

Example 3 Identification of Human ECAT Gene (1) Blast Search of HumanGenomic DNA Database and Human Protein Database

As a result of Blast search, ECAT2-5, 7, 8 genes were found to haveortholog having an amino acid sequence identical in not less than 50%.For ECAT9 gene, too, hECAT9 gene exists as hGDF3 (Caricasole et al.,mentioned above). As regards ECAT1 gene and ECAT6 gene, human orthologcould not be identified.

As a result of BlastP search, there was no publication of base sequenceor amino acid sequence including hypothetical protein, for 3 genes ofhECAT3 gene, hECAT5 gene and hECAT8 gene.

Example 4 Confirmation of Expression of Human Homologous Gene

The ES cell specific expression of ECAT gene in primates was confirmed.

Respective total RNAs derived from 13 kinds of organs of adult human(purchased from Sawady Technology Co., Ltd. or Funakoshi Co., Ltd.),total RNA derived from human mesenchymal stem cell (purchased fromTakara), and total RNA derived from simian ES cell (undifferentiated anddifferentiation induced with retinoic acid, provided by ProfessorNakatsuji of the Institute For Frontier Medical Sciences) were analyzedby the Northern blotting method. The full length cDNA of EST clonecorresponding to hECAT2,4,7,8,9 and hOct3/4 was used as a probe. Whilehybridization was performed in the same manner as in the analysis ofmouse ECAT in the above-mentioned Example 1, the temperature of reactionand washing was set lower (50° C.) so that simian RNA could be detectedusing the human probe. As a result, every gene showed a strong signal inundifferentiated ES cell (FIG. 2). Along with the differentiation of theES cell, signal was dramatically attenuated. While a smear thin signalwas observed in other organs (cells), this is considered to be anonspecific one caused by the lowered temperature of reaction andwashing. From the foregoing results, it has been confirmed that ECATgenes selectively express in ES cells of not only mouse but of primates,as marker genes thereof.

INDUSTRIAL APPLICABILITY

According to the present invention, 9 kinds of ECAT genes specificallyexpressed in mouse ES cell can be newly provided. In addition, humanECAT genes corresponding to 7 kinds out of these 9 kinds can beprovided. Moreover, selective cell markers of ES cell can be obtained bycombining these ECAT genes or fragments thereof. Furthermore, thepresent invention is more effective for a method for selecting ES cellbased on a combination with drug resistance gene, in an attempt tointroduce somatic cell into ES cell-like cell and the like than thesingle use of Oct-3/4 gene or a fragment thereof, and is considered tobe useful in the actual application of a regenerative therapy and thelike.

This application is based on a patent application No. 2001-165927 filedin Japan, the contents of which are hereby incorporated by reference.

1. An antibody that specifically binds to a protein of the following (a)or (b): (a) a protein having an amino acid sequence depicted in SEQ IDNO: 16 or 32; (b) a protein which has an amino acid sequence of (a),wherein one to several amino acids are deleted, substituted or added,and which is specifically expressed in an ES cell.
 2. The antibody ofclaim 1, wherein the antibody specifically binds to a protein having anamino acid sequence depicted in SEQ ID NO:
 16. 3. The antibody of claim1, wherein the antibody specifically binds to a protein having an aminoacid sequence depicted in SEQ ID NO:
 32. 4. The antibody of claim 1,wherein the antibody specifically binds to a protein having an aminoacid sequence depicted in SEQ ID NO: 16, wherein one to several aminoacids are deleted, substituted or added, and which is specificallyexpressed in an ES cell.
 5. The antibody of claim 1, wherein theantibody specifically binds to a protein having an amino acid sequencedepicted in SEQ ID NO: 32, wherein one to several amino acids aredeleted, substituted or added, and which is specifically expressed in anES cell.